Evidence for increased myofibrillar mobility in desmin-null mouse skeletal muscle.

Abstract

Quantitative electron microscopy was used to characterize the longitudinal mobility of myofibrils during muscle extension to investigate the functional roles of skeletal muscle intermediate filaments. Extensor digitorum longus fifth toe muscles from wild-type (+/+) and desmin-null (des -/-) animals were passively stretched to varying lengths, and the horizontal displacement of adjacent Z-disks in neighboring myofibrils (Deltax(myo)) and average sarcomere length (SL) were calculated. At short SL (<2.20 microm), wild-type and desmin-null Deltax(myo) were not significantly different, although there was a trend towards greater Z-disk misalignment in muscles from knockout animals (Deltax(myo) 0.34+/-0.04 microm versus 0.22+/-0.09 microm; P>0.2; means +/- S.E.M.). However, at higher SL (>2.90 microm), muscles from knockout animals displayed a dramatically increased Deltax(myo) relative to wild-type muscles (0.49+/-0.10 microm versus 0.25+/-0.07 microm; P<0.05). The results, which establish a maximum extension of the desmin network surrounding the Z-disk, provide what we believe to be the first quantitative estimation of the functional limits of the desmin intermediate filament system in the presence of an intact myofibrillar lattice. The existence of a limit on the extension of desmin suggests a mechanism for the recruitment of desmin into a network of force transmission, whether as a longitudinal load bearer or as a component in a radial force-transmission system.

Full text

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Desmin, the most abundant intermediate filament protein
in adult striated muscle, plays a critical role in the
organization of the myofibrillar matrix. Desmin connects Z-
disks laterally and, to a lesser extent, longitudinally to one
another as well as to nuclei, mitochondria and costameres
(Georgatos et al., 1987; Granger and Lazarides, 1979, 1982;
Lazarides, 1982; Milner et al., 2000; Richardson et al., 1981;
Tokuyasu et al., 1982; Wang and Ramirez-Mitchell, 1983).
Because of the extensive co-localization of desmin and these
critical elements within the muscle fiber, it is believed that
the intermediate filament network plays the mechanical role
of maintaining proper alignment of cellular structures during
physiological functioning of muscle (Lazarides, 1980; Milner
et al., 1996).
In spite of plentiful information regarding the localization of
desmin filaments throughout the muscle cell, there is scant
evidence for the mechanical function of the intermediate
filament system in skeletal muscle. In ‘ghost’ muscle fibers, in
which the myofibrillar apparatus was chemically extracted,
Wang and Ramirez-Mitchell (1983) measured significant
mechanical load-bearing by intermediate filaments only at very
long sarcomere lengths, over 5µm. This result suggested that
the muscle intermediate filaments may not bear significant
loads at physiological sarcomere lengths, which rarely exceed
3.5µm (Burkholder and Lieber, 2001). However, indirect
evidence suggests that desmin does play an important
functional role in normal muscle. Muscles from desmin-null
(des –/–) mice created via homologous recombination (Li et
al., 1996; Milner et al., 1996) generate a lower isometric stress
(Sam et al., 2000) and exhibit decreased strength (Li et al.,
1997) than muscles from wild-type (+/+) mice. However,
paradoxically, in spite of the ‘weakness’ of these muscles,
the desmin-null muscles appear to be protected from the
mechanical injury that occurs after a bout of eccentric
contractions, even when corrected for the lower stresses
generated by the muscles from knockout animals (Sam et al.,
2000). It is difficult to explain such observations on the basis
of the immunolocalization and ultrastructural data currently
available. Thus, the purpose of the present study was to
quantify the longitudinal mobility of myofibrils during muscle
extension to elucidate further the functional roles of
intermediate filaments in skeletal muscle.
321
The Journal of Experimental Biology 205, 321–325 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
JEB3848
Quantitative
characterize the longitudinal mobility of myofibrils during
muscle extension to investigate the functional roles
ofskeletal muscle intermediate filaments. Extensor
digitorum longus fifth toe muscles from wild-type (+/+)
and desmin-null (des –/–) animals were passively stretched
to varying lengths, and the horizontal displacement of
adjacent Z-disks in neighboring myofibrils (∆xmyo) and
average sarcomere length (SL) were calculated. At short
SL (<2.20µm), wild-type and desmin-null ∆xmyo were not
significantly different, although there was a trend towards
greater Z-disk misalignment in muscles from knockout
animals (∆xmyo 0.34±0.04µm versus 0.22±0.09µm; P>0.2;
means ±
muscles from knockout animals displayed a dramatically
electron microscopy was used to
S.E.M.). However, at higher SL (>2.90µm),
increased
(0.49±0.10µm versus 0.25±0.07µm; P<0.05). The results,
which establish a maximum extension of the desmin
network surrounding the Z-disk, provide what we believe
to be the first quantitative estimation of the functional
limits of the desmin intermediate filament system in the
presence of an intact myofibrillar lattice. The existence of
a limit on the extension of desmin suggests a mechanism
for the recruitment of desmin into a network of force
transmission, whether as a longitudinal load bearer or as a
component in a radial force-transmission system.
∆xmyo
relative to wild-type muscles
Key words: passive strain, electron microscopy, intermediate
filament, force transmission, muscle, mouse.
Summary
Introduction
Evidence for increased myofibrillar mobility in desmin-null mouse skeletal muscle
Sameer B. Shah1, Fong-Chin Su2, Kimberly Jordan1, Derek J. Milner3, Jan Fridén4,
Yassemi Capetanaki3and Richard L. Lieber1,*
1Departments of Orthopaedics and Bioengineering, Veterans Affairs Medical Center and University of California at
San Diego, San Diego, CA 92093, USA, 2Institute of Biomedical Engineering, National Cheng Kung University,
Tainan, Taiwan,3Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA and
4Department of Hand Surgery, Sahlgrenska University Hospital, Göteborg, Sweden
*Author for correspondence (e-mail: rlieber@ucsd.edu)
Accepted 22 November 2001

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Materials and methods
Experiments were performed on the fifth toe muscle of the
extensor digitorum longus (EDL) muscle in two groups of age-
matched young adult mice: wild-type 129/Sv (N=7, 8–12
weeks; Taconic Farms, Germantown, NY, USA) and desmin
homozygous knockout 129/Sv (N=9, aged 8–12 weeks)
(Milner et al., 1996). The EDL of the fifth toe was chosen on
the basis of its distinct origin and insertion tendons, fiber length
homogeneity, unipennate architecture and predominantly fast
fiber-type distribution (Chleboun et al., 1997). Accordingly,
the fifth toe model allows for consistent application of
longitudinal passive loads in a system free from the
confounding effects of fiber rotation or pre-existing
ultrastructural abnormalities, such as those potentially present
in chronically activated desmin-null muscles such as the soleus
or diaphragm (Li et al., 1996; Milner et al., 1996).
All procedures were performed in accordance with the NIH
Guide for the Use and Care of Laboratory Animals and were
approved by the University of California and Department of
Veteran’s Affairs Committees on the Use of Animal Subjects
in Research. Each mouse was anesthetized with a cocktail of
10mgkg–1
ketamine, 5mgkg–1
acepromazine delivered by intraperitoneal injection. The
mouse was then killed by intracardiac injection of concentrated
sodium pentobarbital. The hindlimbs were skinned and
transected below the hip, leaving the entire knee joint intact,
and placed for further dissection (within 15min of death) into
a Ringer’s solution (adjusted to pH7.5) composed of 137 mmol
l–1NaCl, 5 mmoll–1KCl, 1 mmoll–1NaH2PO4, 2 mmoll–1
CaCl2, 1 mmoll–1MgSO4and 11 mmoll–1glucose containing
10mgl–1curare. Each muscle was then dissected, passively
stretched from approximately 100 to approximately 150% of
slack muscle length to generate a range of sarcomere lengths
and tied to a wooden applicator.
Stretched muscles were submerged into phosphate-buffered
Karnovsky’s fixative (6% buffered glutaraldehyde plus
formaldehyde), in which they were allowed to incubate
overnight at 4°C. Specimens were washed three times in cold
(4°C) sodium cacodylate buffer (0.1moll–1adjusted to pH7)
and then further fixed in 2% osmium tetroxide for 1h at room
temperature (23°C). After three buffer washes of 5min each,
muscles were dehydrated in a graded ethanol series and
propylene oxide. Specimens were then cut into approximately
equal-sized pieces, embedded in Scipoxy 812 Resin (Energy
Beam Sciences, Agawam, MA, USA) and oriented to enable
longitudinal sectioning. Micrographs were photographed from
longitudinal sections of deep and superficial regions
(approximately 150µm2per micrograph) randomly distributed
through the fiber (five micrographs for each depth, giving 10
micrographs per muscle). The sections were confirmed to be
in perfect longitudinal alignment by tracking the myofibrils
(maximum diameter approximately 0.7µm) across the entire
micrograph (maximum length approximately 14µm) without
observing myofibrils shifting into and out of the section plane.
The end points of all Z-disks from each micrograph were
digitized (Fig. 1), allowing the quantification of the horizontal
rompum and 1mgkg–1
displacement of adjacent Z-disks (∆xmyo; 45–80 measurements
per micrograph) and the calculation of sarcomere length (SL;
five measurements per micrograph).
The contributions of myofibrils, sarcoplasmic reticulum and
mitochondria to total cell volume were quantified using
stereological techniques on each of the longitudinal sections
S. B. Shah and others
Fig. 1. Electron micrographs of the extensor digitorum longus fifth
toe muscle from wild-type (A) or desmin-null (B) animals fixed
under conditions of passive loading, displaying the method for
digitizing Z-disk endpoints to yield horizontal Z-disk displacement
(∆xmyo) and sarcomere length (SL). Note the greater stagger in
adjacent Z disks in muscle from knockout versus wild-type animals.
For the frames displayed (approximately one-fifteenth the area of a
full micrograph), for (A), ∆xmyo=0.09µm and SL=3.57µm, for (B),
∆xmyo=0.58µm and SL=3.60µm. The calibration is identical for both
micrographs. Scale bar, 1µm.

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Myofibrillar mobility in desmin-null muscles
described above. To prevent pattern recognition or bias during
micrograph analysis, sets of micrographs from both superficial
and deep regions of muscles from wild-type and desmin-null
mice were catalogued and merged in random order. In addition,
the technician responsible for analysis did not know the
hypothesized results, fiber depth or genotype of the
micrographs.
A 10×10 grid of 15mm squares was printed on a
transparency, and coordinates were numbered on each side.
The grid was placed at a fixed point on each micrograph, and
the structure at each one of the 100 intersecting test points was
categorized as myofibril,
reticulum (SR) or ‘other,’ where ‘other’ represented
extramyofibrillar structures that could not definitively be
categorized as either mitochondria or SR. At test points lying
on borders between structures, the component visible in the
upper left corner of the intersection was credited to the point
in question. Finally, micrographs were resorted into their
original groups upon completion of point counting, and the test
point fraction of each structure was tabulated, corresponding
to the volume density, or volume fraction, of each structure
(Weibel, 1980).
An unpaired t-test was used to determine whether horizontal
Z-disk displacement and/or
significantly different in deep and superficial regions of muscle
fibers, and linear regression was used to measure the
association between ∆xmyo and SL in response to
passive stretch. The slopes for data from muscles of
wild-type versus knockout animals were compared by
analysis of covariance (ANCOVA). Differences in
misalignment at short (SL<2.20µm) and long
(SL>2.90µm) sarcomere lengths, corresponding to
strains resulting in insignificant and significant passive
loads, respectively, were compared by one-way
analysis of variance (ANOVA) (Statview 5.0, Abacus
Concepts, Inc., Berkeley, CA, USA). The range of
sarcomere lengths analyzed also corresponds to the
shortest and longest operating sarcomere lengths for
the mouse EDL (James et al., 1995). Values obtained
from point counting were compared using a two-way
ANCOVA, with genotype (wild-type versus desmin-
null) and depth (superficial versus deep) as grouping
factors and sarcomere length as the covariate.
mitochondria, sarcoplasmic
sarcomere length were
Results
Horizontal Z-disk displacement, ∆xmyo, in deep and
superficial regions of EDL fifth toe muscles from
knockout and wild-type animals was plotted against
sarcomere length to characterize myofibrillar mobility
with increasing sarcomere strain (Fig. 2). There were
no significant differences in ∆xmyo or SL between
superficial and deep regions of muscle fibers within
muscles of knockout or wild-type animals (unpaired t-
test, SL, P=0.95; ∆xmyo, P=0.92). Consequently, the
data were pooled from the superficial and deep regions
within each muscle type for further statistical analysis. At short
SL (<2.20µm), wild-type and desmin-null ∆xmyo values were
not significantly different, although there was a trend towards
greater Z-disk misalignment in muscles from knockout animals
(0.34±0.04µm versus 0.22±0.09µm, P>0.2; means ± S.E.M.).
However, at higher SL (>2.90µm), muscles from knockout
animals displayed an increased ∆xmyorelative to muscles from
wild-type animals (0.49±0.10µm versus
P<0.05).
The observation that there is a difference in ∆xmyobetween
myofibrils from knockout and wild-type animals with
increasing sarcomere strain was supported by the fact that the
regression slope of the ∆xmyo versus SL relationship was
significantly different from zero in the specimens from
knockout animals (P<0.005) but not in specimens from wild-
type animals (P>0.4). In addition, ANCOVA revealed a
significant difference between the slopes of these relationships
(P<0.05). Finally, the coefficients of determination (r2) and
regression slopes (m) were both dramatically higher in the
specimens from knockout animals compared with wild-type
animals (wild-type, m=0.03, r2=0.10; knockout, m=0.15,
r2=0.81). Taken together, a significant difference in the ∆xmyo
value between genotypes was seen as a function of sarcomere
length in this experiment.
No significant differences in myofibrillar, mitochondrial or
SR content between muscles from wild-type or desmin-null
0.25±0.07µm,
/
/
4.03.53.02.52.01.5
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Wild type
Desmin null
Sarcomere length (µm)
Z-Disk displacement, ∆xmyo (µm)
Fig. 2. Horizontal Z-disk displacement (∆xmyo) plotted versus sarcomere
length (SL) for extensor digitorum longus fifth toe muscles of wild-type
(circles) and desmin-null (squares) animals. Superficial regions are shown
with open symbols while deep regions are shown with filled symbols. Note
that ∆xmyo increases to a greater extent as SL increases in muscles from
knockout compared with wild-type animals. Error bars represent ±1 S.E.M. in
either the ∆xmyoor SL direction (N=7 wild-type muscles and N=9 desmin-null
muscles). Superficial and deep regions of each muscle were sampled from 10
micrographs per region. The regression slope of the ∆xmyo versus SL
relationship was significantly different from zero in the specimens from
knockout animals (P<0.005) but not in the specimens from wild-type animals
(P>0.4), and analysis of covariance (ANCOVA) revealed a significant
difference between the slopes of these relationships (P<0.05).

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animals were observed (P>0.6), nor were differences seen as
a function of depth (P>0.6) (Table 1). Sarcomere length was
noted to be a significant covariate (P<0.01) from ANCOVA,
indicating that these values were sensitive to the fixation
length.
Discussion
This study provides what we believe to be the first
quantification of the limits of functional extension of the
desmin intermediate filament system in the presence of an
intact myofibrillar lattice. Inspection of electron micrographs
revealed that the sarcomeres themselves, within a single
myofibril, are phenotypically normal, exhibiting the
characteristic Z-disk, I-band and A-band morphology (Fig. 1).
However, the increase in Z-disk misalignment with increasing
sarcomere length in muscles from desmin-null specimens
suggests that desmin plays a role in organizing intact
myofibrils laterally during mechanical loading by tethering
adjacent Z-disks. It is also possible that the absence of
longitudinal desmin linkages may result in a more random and
misaligned myofibrillar network. However, this seems unlikely
since it would probably also result in a large sarcomere length
variation longitudinally, which was not the case (see horizontal
error bars in Fig. 2).
The maximum ‘tethering radius’ (a term coined to describe
the maximum longitudinal extension of the desmin network
surrounding a Z-disk) is estimated to be approximately
0.32µm from the Z-disk displacement in muscles from wild-
type animals at high passive strain. This value remained
relatively constant as a function of sarcomere length. In
contrast, in knockout animals, the value of ∆xmyoincreased as
a function of sarcomere length and, thus, an upper value for
tethering radius cannot be readily inferred (Fig. 2). The
existence of a limit on the extension of desmin in wild-type
animals suggests a mechanism for the recruitment of desmin
into a network of force transmission, whether as a longitudinal
load bearer or as a component in a radial force-transmission
system. Specifically, desmin could act as a highly compliant
or unloaded spring at low extensions, but at increasing
extension would exhibit the properties of an extremely stiff
spring or, quite possibly, a rigid linkage. Details of this
relationship could be elucidated by characterizing the
mechanical properties of the desmin filaments themselves. In
addition, such a network of filamentous connections, which
integrates the entire cytoskeleton, could play an important role
in strain-mediated signal transduction, such as that suggested
by a study of endothelial cells (Maniotis et al., 1997).
Analogous studies could be envisioned in muscle, in which
force- and length-transducing mechanisms could be carefully
studied in wild-type or desmin-null mice.
The absence of a tethering radius in muscles from knockout
animals argues against other intermediate filaments providing
a substitute function for absent desmin. These proteins include
those associated with the M-line, such as skelemin (Price,
1987; Price and Gomer, 1993). These filaments either have a
fracture stress less than the passive stress imposed at high
strains, and are therefore damaged during increased extension,
or are extremely compliant at high strains, and therefore play
a minimal role in the structural organization of myofibrils.
Finally, from a design standpoint, the absence of a limit to
∆xmyo in desmin-null muscles provides further evidence
against any upregulation
intermediate filament proteins in the knockout system that
would mimic desmin function, such as paranemin, synemin or
plectin (Carlsson et al., 2000), since, if these proteins were
dramatically upregulated, one might expect less mobility in the
myofibrils of muscles from desmin-null animals.
Differences in Z-disk displacement between muscles from
wild-type and knockout animals may explain the observation
of the lower isometric stress generated by desmin-null
muscles as well as their reduced susceptibility to injury that
we recently reported (Sam et al., 2000). However, Z-disk
displacement was measured in the previous study only from
specimens at slack length. Thus, it was not clear whether the
differences were permanent, fixed differences between
muscles or whether they suggested underlying differences in
interconnections between adjacent myofibrils. The present
data support the idea of differences in interconnections
between muscles of different genotypes. On the basis of the
increased mobility observed in desmin-null muscles, sliding
of adjacent myofibrils could result in inefficient force
transmission due to energy dissipation (explaining the
lower isometric stress). Using similar logic, mechanically
uncoupling myofibrils during mechanical loading of a muscle
fiber could serve a protective role (explaining the decreased
muscle injury). It is conceivable that damaged myofibrils
without a surrounding desmin intermediate filament lattice
(Lieber et al., 1996) would have an opportunity to repair
themselves prior to re-integrating themselves into the load-
bearing network. During this myofibrillar repair, muscle
function would not be completely compromised since other
sarcomeres could transmit force normally. It does not appear
that the increase in myofibrillar mobility or decrease in force-
generating ability (Sam et al., 2000) in muscles from desmin-
knockout animals is due to inherent differences in myofibrillar
content (Table 1). Note, however, that it is possible that
stereology of transverse muscle sections may reveal
of functionally analogous
S. B. Shah and others
Table 1. Volume per cent of structures in muscle samples
Wild typeDesmin null
Deep
(%)
Superficial
(%)
Deep
(%)
Superficial
(%)Structure
Myofibril
Mitochondria
Sarcoplasmic
reticulum
78.2±1.7
4.9±0.7
7.9±1.8
78.6±1.5
4.0±0.6
9.7±1.7
82.9±2.7
4.3±1.1
6.2±1.6
76.0±2.1
5.1±1.2
6.5±2.1
Values are means ± S.E.M.; N=10 micrographs per muscle.
No significant differences were observed between genotypes or
depths.

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Myofibrillar mobility in desmin-null muscles
differences in the density of structures involved in the
excitation–contraction coupling process.
Finally, the observation of increased Z-disk mobility in
desmin-knockout mice at SL well below 5.0µm, i.e. lengths at
which Wang and Ramirez-Mitchell (1983) reported significant
passive load-bearing ability in ‘ghost’ fibers, suggests that the
role of intermediate filaments should be re-examined in a
system free from the confounding geometrical effects of
sarcomere protein extraction. In particular, it is possible that
the collapse of the intermediate filament system as a result
of myofibrillar extraction altered the mechanical boundary
conditions of the intermediate filament lattice during testing,
thereby significantly affecting the mechanical properties
measured.
This work was supported by the Department of Veterans
Affairs and NIH grant 40050 and the Swedish Medical
Research Council. We thank Heather Ross for excellent
technical assistance and Professor Odile Mathieu-Costello
(UC San Diego) for assistance in designing the stereology
protocol.
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